Oral and Poster Abstracts
Basic Science and Clinical Practice in Blood Transfusion: Poster II
Hall A3/A4 (Orange County Convention Center)
Poster Board III-140
High titer inhibitory antibodies (Abs) to Factor VIII (FVIII) generated in response to FVIII replacement therapy can render the therapy ineffective in hemophilia A (HA) patients. Abs to FVIII contain serine protease-like nucleophilic sites that react covalently with electrophilic phosphonate diester groups placed on the Lys side chains of FVIII (E-FVIII), resulting in restoration of the ability of exogenous FVIII to act as a cofactor for blood coagulation (Planque et al, J Biol Chem 2008; 283, p11876-11886). Irreversible inactivation of Abs by E-FVIII could potentially permit administration of FVIII for treatment of acute bleeding episodes in HA patients with high titer inhibitory antibodies. Another favorable effect of E-FVIII is its ability to block the production of anti-E-FVIII Abs by cultured memory B cells (Smith et al., J Thromb Haemost 2007; 5 Supplement 2: O-M-063). Here we report the resistance of E-FVIII to proteolytic clearance, a property predicted to enable prolonged blockade of pathogenic anti-FVIII immunity. E-FVIII was degraded by the serine proteases thrombin, trypsin and chymotrypsin at rates 100, 29 and 23-fold less rapidly, respectively, compared to FVIII, determined by electrophoretic generation of protein fragments. The effect of E-FVIII on thrombin catalyzed VAL-PRO-ARG-aminomethylcoumarinamide hydrolysis followed classical irreversible inhibitory kinetics, consistent with covalent bonding of the phosphonate group to the thrombin active site. Thrombin affinity (Ki) for E-FVIII and the rate of covalent bond formation (k2), respectively, were 1331 nM and 1.8 min-1. E-FVIII resistance to thrombin should reduce its consumption in the coagulation pathway, increasing its availability for the reaction with anti-FVIII Abs. FXa, an enzyme that contributes in the metabolic clearance of FVIII from blood, also degraded E-FVIII more slowly compared to FVIII. The half-life of E-FVIII in human serum and human plasma in vitro was substantially superior to that of FVIII. Only weak binding of E-FVIII to human von Willebrand Factor (VWF) was detected, suggesting that stabilizing interactions with VWF are not the reason for increased E-FVIII resistance to the proteases. E-FVIII did not prolong the time to clot formation determined by the APTT test and did not inhibit FXa generation in the chromogenic Coamatic test. Therefore, E-FVIII does not cause a global inhibition of the serine protease pathways required for blood coagulation. The reaction model entails covalent bonding of the E-FVIII phosphonate if productive noncovalent contact with the enzyme is established. As the E-FVIII is present at limiting concentrations (<Ki), generalized protease inhibition does not occur. These data suggest that clearance of E-FVIII in vivo by blood-borne proteases should be limited, potentially affording a longer half-life and prolonged blockade of pathogenic antibody effects. Reduced E-FVIII digestion by intracellular proteases may also attenuate the presentation of pathogenic FVIII antigenic epitopes to T cells, thereby, reducing the production of inhibitory anti-FVIII antibodies.
Disclosures: Planque: Covalent Immunology Products: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Escobar: Covalent Immunology Products: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Nishiyama: Covalent Immunology Products: Consultancy, Equity Ownership, Patents & Royalties. Paul: Covalent Immunology Products: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.
*signifies non-member of ASH